A Specific and Covalent JNK‐1 Ligand Selected from an Encoded Self‐Assembling Chemical Library

Abstract

We describe the construction of a DNA-encoded chemical library comprising 148'135 members, generated through the self-assembly of two sub-libraries, containing 265 and 559 members, respectively. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed against JNK1, a kinase containing a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compounds were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mM), used to simulate intracellular reducing conditions. By contrast, derivatives of the individual building blocks were not able to covalently modify JNK1 in the same experimental conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site. A distinction is often made between "single pharmacophore" and "dual pharmacophore" chemical libraries. In the first case, individual compounds (no matter how complex) are coupled to a DNA fragment, which serves as amplifiable identification barcode. In the second case, two building blocks are simultaneously connected to the extremities of complementary DNA strands, thus enabling the formation of combinatorial libraries by the self-assembly of oligonucleotide conjugates. [3] We have recently described a strategy for the encoding of dualpharmacophore libraries (also called "encoded self-assembling chemical libraries", or ESAC libraries), which was compatible with library decoding procedures, based on high-throughput DNA sequencing. [4] ESAC libraries may facilitate the identification of synergistic building blocks, which recognize adjacent pockets on target proteins of interest. These chemical moieties need to be subsequently connected through a suitable chemical linker, in order to display protein binding in the absence of DNA. [4,5,6] Encoded chemical libraries have previously been used for the discovery of covalent protein binders. For example, Nicolas Winssinger and coworkers previously reported the identification of reversible and irreversible covalent binders of bromodomain, [7] kinases, [8] proteases and phosphatases [9] from both DNA and PNA encoded chemical libraries. Recently, Cuozzo et al. 4 published the discovery of a potent covalent inhibitor of Bruton's tyrosine kinase (BTK), with picomolar IC 50 value from a single DNA encoded library. [10] Here, we describe the construction of a DNA-encoded self-assembling chemical library, formed by combination of 265 x 559 building blocks, yielding an ESAC library with 148'135 members. The library was designed to incorporate building blocks, which were potentially capable of forming covalent interactions with ce...

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues

“…[26] DNA-encoded libraries have previously been used to discover irreversible covalent inhibitors. [11,19] Moreover,DNA structures have been used to probe the magnitude of affinity gain by polydentate engagement with multivalent protein target. Encoded self-assembling chemical (ESAC) libraries are particularly attractive for the discovery of covalent inhibitors since DNAc onjugates can be individually purified to homogeneity,t hus preserving the integrity of reactive functional groups.…”

“…Figure 1A depicts how Schiff-base formation for benzaldehyde derivatives may be in competition with the hydration of the aldehyde moiety and how these equilibria can be perturbed by neighboring functional groups.T he two reactions can be monitored and quantified using 1 HNMR methods ( Figure 1B). [11,12] In this work, we initially assessed the contribution of benzaldehyde derivatives to protein binding for various ligands,u sing chemical derivatives of short (12-bp) locked nucleic acids (LNAs) (Figure 2A). The ortho-formyl-phenylboronic acid (14) moiety,which had been used for the generation of reversible covalent Mcl-1 inhibitors, [3] exhibited a K D value of 17 mm, while phthaldialdehyde (16)s trongly reacted to form an irreversible 1-isoindolinone adduct.…”

Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA‐Encoded Self‐Assembling Chemical Libraries

“…For practical applications,i ti sn ecessary to link these ligand pairs into discrete organic molecules,d evoid of any nucleic acid component. [22][23][24] ESAC technology represents one experimental modality for the construction and screening of DNA-encoded chemical libraries. The procedurey ieldedas et of small organic ligands, the best of which exhibited ad issociation constanto f9 .9 nm,a sm easured in solution by fluorescencepolarization.…”

“…[8][9][10][11][12][13][14][15][16][17][18][19][20][21] We have previously reportedt hat the discovery of ligand pairs that synergistically bind to at arget protein can be facilitated by the use of DNA-encoded self-assemblingc hemical libraries (ESAC libraries). [50] We have subsequently expanded the size and diversity of the ESAC library [24] and used it for new AGP selections, incorporating analogues of the originally discovered binding pairs. Here, we describe ac hemical strategy that expeditest he process of fragment-linking, the measurement of binding affinitiesa nd the identification of highly potent small-molecule binders.…”

“…[1][2][3][4][5][6][7] Acommon methodology to increase potencyi nF BDD relies on the linking of fragment pairs via appropriate scaffolds. [22][23][24] ESAC technology represents one experimental modality for the construction and screening of DNA-encoded chemical libraries. [22][23][24] ESAC technology represents one experimental modality for the construction and screening of DNA-encoded chemical libraries.…”